Chemoautotrophic growth of Rhodopseudomonas species with hydrogen and chemotrophic utilization of methanol and formate

A chemolithoautotrophic type of metabolism, which was hitherto unknown for purple nonsulfur bacteria, was demonstrated by growth experiments using Rhodopseudomonas capsulata Kb1 and Rhodopseudomonas acidophila 10050. These strains were able to grow in a mineral medium in the dark at the expense of H₂, O₂, and CO₂. A minimum doubling time of 9 h was obtained for R. capsulata under an atmosphere containing less than 15% oxygen; higher oxygen concentrations suppressed autotrophic but not chemoorganotrophic growth. Oxygen sensitivity of chemoautotrophically growing cells of R. acidophila was even more pronounced, whereas cells growing chemotrophically on methanol almost tolerated the oxygen concentration of air. Highest oxygen sensitivity of growth of R. acidophila was observed with formate as substrate. The growth yield of cultures grown semiaerobically in the dark on methanol was 0.23 g dry cell material per g methanol consumed.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection,helminthiasis, thrombosis and neuropharmacological disorders

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds’ phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 μg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2′-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.     

甲醇的生物利用与转化*

甲醇作为一种来源广泛、价格低廉、还原度高的非粮原料有望成为下一代生物制造的关键原料。利用合成生物学技术构建能够高效利用甲醇的重组微生物以实现从甲醇到高值化学品的生物转化已成国内外研究热点,但由于甲醇代谢过程的特殊性及复杂性,目前人工设计的甲基营养菌还难以实现以甲醇为唯一碳源进行生长及产物合成。基于对天然甲基营养菌甲醇代谢过程的分析,从甲醇脱氢酶的筛选与改造、甲醛同化途径的重构与优化、甲醇到化学品的生物转化几个方面对合成型甲基营养菌的构建策略及面临的挑战进行总结与分析,以期为今后合成型甲基营养菌的人工设计和利用提供一定的借鉴。     

无花果果皮中花青素的提取工艺的建立

本研究使用单因素方法考察了无花果(Ficus carica L.)果皮中花青素的最佳提取条件,并考察了7种参数对花青素提取率的影响。参数设置如下:溶剂性质(水,甲醇,乙醇和丙酮)、提取次数(1~3次)、固液比(1/50,1/100,1/150和1/200)、提取时间(60 min,120 min,180 min和240 min)、甲醇浓度(0,20%,40%,60%,80%和100%)、酸类型(盐酸,乙酸,柠檬酸和酒石酸)和酸浓度(0,1%,2%,5%和10%)。使用pH-示差法测量无花果果皮中单体花色素的含量。研究显示,无花果果皮中花青素的最佳提取条件为:溶剂为甲醇溶剂,提取次数为2次,固液比为1/100,提取时间为180 min,甲醇浓度为80%,酸类型为柠檬酸,柠檬酸浓度为5%。该最佳提取条件下的花青素的提取率达到最高(345.62 mg/100g DS)。     

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.     

Quantitative assessment of methyl‐esterification and other side reactions in a standard propionylation protocol for detection of histone modifications

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS‐based analysis, chemical derivatization of N‐terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl‐esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl‐esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in‐depth quantitative analyses of methyl‐esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl‐esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.     

Long-term adaptation of methanol-fed thermophilic (55 °C) sulfate-reducing reactors to NaCl

A laboratory-scale upflow anaerobic sludge bed (UASB) reactor was operated during 273 days at increasing NaCl concentrations (0.5–12.5 g NaCl l^–1) to assess whether the stepwise addition of the salt NaCl results in the acclimation of that sludge. The 6.5-l thermophilic (55 °C), sulfidogenic [a chemical oxygen demand (COD) to SO₄^2– ratio of 0.5] UASB reactor operated at an organic loading rate of 5 g COD l^–1 day^–1, a hydraulic retention time of 10 h and was fed with methanol as the sole electron donor. The results show that the adaptation of the thermophilic, sulfidogenic methanol-degrading biomass to a high osmolarity environment is unlikely to occur. Sulfide was the main mineralization product from methanol degradation, regardless of the NaCl concentration added to the influent. However, sulfide production in the reactor steadily decreased after the addition of 7.5 g NaCl l^–1, whereas acetate production was stimulated at that influent NaCl concentration. Batch tests performed with sludge harvested from the UASB reactor when operating at different influent salinities confirmed that acetate is the main metabolic product at NaCl concentrations higher than 12.5 g l^–1. The apparent order of NaCl toxicity towards the different trophic groups was found to be: sulfate-reducing bacteria > methane-producing archaea > acetogenic bacteria.